Adeno-associated virus serotype 2 (AAV2) is a human parvovirus that is being developed as a gene therapy vector. AAV2 requires co-infection with a helper virus, usually an adenovirus or herpesvirus, for efficient productive infection. In the absence of helper virus, AAV2 DNA can integrate into the host genome with a strong preference (70%-90% of integration events) for a 4 kb region of human chromosome 19, designated AAVS1 (the only example of site-specific integration in a mammalian virus system). This ability to specifically integrate also contributes to AAV2's attractiveness as a vector for gene therapy, since this could potentially limit the dangers associated with insertional mutagenesis. During AAV2 replication, the Rep68 and Rep78 proteins (Rep68/78) of AAV2 make a site- and strand-specific nick at the terminal resolution site (trs) within the stem of the hairpin structure formed by the inverted terminal repeats (ITRs) of AAV2 DNA. The nicking activity of Rep68/78 is also essential to the preferential integration process and a nicking site has been identified within AAVS1. The current nicking model suggests that the strand containing the nicking site is separated from its complementary strand prior to nicking. In AAV serotypes 1 through 6, the nicking site is flanked by a sequence that is predicted to form a stem-loop with standard Watson-Crick base pairing. The region flanking the nicking site in AAVS1 (5'-GGCGGCGGT/TGGGGCTCG -3' [slash indicates nicking site]) lacks extensive potential for Watson-Crick base pairing. We therefore performed an empirical search for a stable secondary structure. By comparing the migration of radiolabeled oligonucleotides containing wild-type or mutated sequences from the AAVS1 nicking site to appropriate standards, on native and denaturing polyacrylamide gels, we have found evidence that this region forms a stable secondary structure. Further confirmation was provided by circular dichroism analyses. We identified six bases that appear to be important in forming this putative secondary structure. Mutation of five of these bases, within the context of a double-stranded nicking substrate, reduces the ability of the substrate to be nicked by Rep78 in vitro. Four of these five bases are outside the previously recognized GTTGG nicking site motif and include parts of the CTC motif that has been demonstrated to be important for integration targeting. These results may further help to explain the specificity of AAV2 integration. Different AAV types use different cellular receptors and vary in their tissue tropisms. Gene therapy vectors based on AAV types other than type 2 may therefore be more useful than AAV2 for specific gene therapy applications. However, current AAV replication and packaging systems use the rep gene of AAV2. We therefore tested the ability of AAV2 Rep78 to cut at the terminal resolution sites within the ITRs of two novel AAV types (AAV8 and AAV rh.39) recently isolated by our collaborators at the University of Pennsylvania. AAV2 Rep78 efficiently nicked the AAV rh.39 5' and 3' ITRs, as well as the AAV8 3'-ITR. To our surprise, no evidence of nicking was observed for the AAV8 5'-ITR. The AAV8 5'-ITR does however have a slightly different sequence than the AAV8 3'-ITR.